Method for the diagnosis of Helicobacter pylori infection, and a diagnostic kit for performing the method

ABSTRACT

The method for the diagnosis of  Helicobacter pylori  infection by the oral administration of defined amounts of  13 C-labeled urea and examination for  13 C content of blood samples removed at a defined time is effected by a) removing from 0.1 to 0.6 ml of capillary blood from the finger or ear lob of a patient or venous blood of a patient, in both cases with an empty stomach before the beginning of the test; 
         b) administering an exact amount of from 10 to 50 mg of  13 C-urea in aqueous solution with a pH value of 2 to 4 to the patient; c) again removing capillary or venous blood exactly after 10 to 15 min from the administration; and    d) determining the  13 C content of the blood samples by isotope ratio mass spectrometry (IRMS), and deducing the presence of  Helicobacter pylori  from the increase of the  13 C values.

The present invention relates to a method for the diagnosis of Helicobacter pylori infection by the oral administration of defined amounts of ¹³C-labeled urea and examination for ¹³C content of blood samples removed at a defined time.

The previously usual and mostly performed method for the diagnosis of Helicobacter pylori infection is the ¹³C-urea respiration test. This method has been described in detail in EP-A-0 253 927. This test has the disadvantage, that it cannot be applied for children below 3 years and adults suffering from breathinsufficiency like asthma.

Rex Moulton-Barrett et al., The American Journal of Gastroenterology, Vol. 88, 1993, pages 369 to 374, describe a method in which ¹³C-labeled hydrogencarbonate is determined in the serum upon oral administration of ¹³C-labeled urea. In this test, 5 mg/kg of ¹³C-labeled urea was administered to the patient, and blood samples of 3 ml each were taken and examined after 15, 30, 60, 90, 120 and 180 min. This method has been further examined and described by Mark J. Kim et al. in Gastroenterology 1997, 113: 31-37, W. D. Chey et al. in The American Journal of Gastroenterology, Vol. 94, 1999, pages 1522 to 1524, Alan F. Cuttler et al. in The American Journal of Gastroenterology, Vol. 94, 1999, pages 959 to 961, and has resulted in the introduction of a test by the company Metabolic Solutions Inc., Nashua, N.H. In this test, without determination of the zero value and upon administration of a high-fat test meal “Ensure”, 125 mg of ¹³C-urea is administered, and the ¹³C content of 3 ml of blood is determined after 30 min. However, due to the low accuracy and precision and due to the very high price of the necessary amount of ¹³C-urea and the relatively high test quantity of blood, this test was not successful economically, so that the respiration test remained the mostly used test method despite of all its disadvantages.

It has been the object of the invention to provide a method for the diagnosis of Helicobacter pylori infection with as low as possible an amount of ¹³C-urea and as low as possible a blood quantity for the examination of the ¹³C content, which method establishes the diagnosis of Helicobacter pylori infection more simply, more inexpensively, more accurately and more quickly.

This object is now achieved by removing from 0.1 to 0.6 ml of capillary blood from the finger or ear lob of a patient or venous blood, in both cases with an empty stomach before the beginning of the test, administering an exact amount of from 10 to 50 mg of ¹³C-urea in aqueous solution with a pH value of 2 to 4 to the patient, again removing capillary or venous blood exactly after 10 to 15 min from the administration, and determining the ¹³C content of the blood samples by isotope ratio mass spectrometry (IRMS), and deducing the presence of Helicobacter pylori from the increase of the ¹³C values.

Even when 0.6 ml of capillary blood and 50 mg of ¹³C-urea is used, this method is clearly superior to the previous method, all the more so since the accuracy of the method is enormously increased by determining the starting value, and the method is significantly abbreviated for the patient by the removal of a second blood sample already after 10 to 15 min. In elder patients, the recovery of capillary blood sometimes involves difficulties. In such cases, the method can also be performed with the same small amount of venous blood.

Of critical importance in the method according to the invention is the omission of the high-fat test meal. Instead, the labeled urea is administered in an aqueous solution with a pH value of from 2 to 4. This can be done, for example, either by adjusting the aqueous solution of the labeled urea by means of a non-volatile, pharmacologically acceptable organic acid to a pH value of from 2 to 4, or by adding a pack of a solid pharmacologically acceptable organic acid to the freshly prepared solution. Citric acid has been found particularly suitable. However, in principle, other organic acids of that kind, such as ascorbic acid, are also capable of adjusting the desired pH value. It is also possible to achieve this low pH value by the use of orange juice, grapefruit juice or sour apple juice.

The determination of the ¹³C content in the blood samples can be effected, for example, by adding a strong non-volatile acid, such as phosphoric acid, which is capable to release the carbon dioxide in a gaseous form from the blood sample, so that it can be measured with an IRMS device.

However, it is also possible to recover serum from the blood samples, to remove high molecular weight components from the serum sample by means of suitable filters, and to determine the ¹³C content in the remaining liquid by a preliminary elemental analysis followed by isotope ratio mass spectrometry.

By comparative examinations, it was established that the detection of a Helicobacter pylori infection in a respiratory test is positive when the value of ¹³C in the respiratory air is about 4‰ above the starting value. In contrast, the method according to the invention is capable of detecting Helicobacter infections when the difference of the ¹³C content is as low as 2.0‰. It is obvious that the substantially lower amount of the expensive ¹³C-urea, on the one hand, and the quickening of the time of the second sampling from 30 min to 10 min are of significant importance to both the patients and the physicians. In addition, the removal of a maximum of 0.6 ml of capillary or venous blood is significantly simpler and more convenient than the removal of 3 ml of venous blood in the test of the Metabolic Solutions Inc. As compared to this test, above all, the significantly higher accuracy and precision is of critical importance since the starting value is determined for each patient rather than using the average values, which are accompanied by considerable variations and influenced by the different foods taken up by the subjects.

The diagnostic kit according to the invention for performing the method preferably consists of an acidic aqueous solution having a pH value of from 2 to 4 and containing exactly from 10 to 50 mg of ¹³C-urea, a patient instruction sheet, two sample vessels for receiving the blood samples, and optionally a blood-sampling device. It is decisive that the kit contains an exaxt amount of ¹³C-urea. A kit for children may contain less than for adults in the over all range of 10 to 50 mg ¹³C-urea. Also the time difference between the two removals of blood should be exactly measured. Different kits, however, may be run in the over all time range of 10 to 15 min.

Another preferred embodiment comprises instead of the ready acidic solution a container for urea with exactly from 10 to 50 mg of ¹³C-urea and a pack of a solid pharmacologically acceptable organic acid, such as citric acid. Normally, 2 g of citric acid in 200 ml of water or 200 ml of orange juice, grapefruit juice or sour apple juice is suitable for dissolving from 30 to 50 mg of ¹³C-urea. Especially for children, the amount of ¹³C-urea can be further decreased down to 10 mg.

The blood samples preferably are taken up in commercially available sample vessels for receiving blood samples, for example, Vacutainer®. Preferably, they may already contain the necessary amount of concentrated phosphoric acid, so that the CO₂ is immediately released from the blood sample. In principle, however, it is also possible to add a strong non-volatile acid later to the sample vessel for receiving the blood sample.

The determination of the ¹³C content is then effected by IRMS directly from the gas phase. An increase of the ¹³C content of as low as 2‰ after 10 min indicates infection with Helicobacter pylori.

Alternatively, serum may also be withdrawn from the blood sample after a short settling period. All macromolecules and especially the lipids can be removed from this serum sample by ultrafiltration. The remaining liquid is then first subjected to elemental analysis by combustion, and the CO₂ released thereby is again examined by IRMS for the isotope ratio ¹³C/¹²C. In this test method, infection with Helicobacter pylori can be detected at an increase of the ¹³C content of as low as from 1 to 1.5‰.

The higher sensitivities and accuracies of the test method achieved according to the invention can be explained afterwards by the fact that the ¹³C content of the labeled urea is to a lesser extent diluted with other carbon sources. This dilution effect is strongest in the respiratory air test and least in the examination of serum samples from which macromolecular molecules are removed by ultrafiltration. Nevertheless, the method in which CO₂ is released from the blood sample by a strong non-volatile acid and determined in the gas phase is already excellently suitable for achieving the object of the present invention.

When the method according to the invention is performed in practice, the blood tests are executed by the physician before and from 10 to 15 min after administration of the ¹³C-urea. The Vacutainers® can be collected and sent to a central laboratory where the determination of the ¹³C contents is effected. From these results fed-back to the physician, he can diagnose whether or not there is an infection with Helicobacter pylori.

In principle, all highly sensitive isotope mass spectrometers available on the market can be used for the IRMS determination; however, due to their high price, they can be set up only in central laboratories. Already for clinics, investment in such devices cannot be expected. In contrast, the collecting and analyzing of samples in central laboratories does not involve any problems today in terms of logistics and prices, and the same applies to the back transmission of analytical data to the submitting physician or the submitting clinic.

The same is true of the determination method in the serum from which high molecular weight components have been removed by ultrafiltration. The preliminary elemental analysis by combustion can be performed in central laboratories without problems. The recovery of serum from capillary or venous blood and ultrafiltration can be performed without problems at least in clinics. For the actual determination of the ¹³C content, 20 μl of serum is sufficient and will yield telling results already for an increase of from 1 to 1.5‰ of ¹³C. 

1-5. (canceled)
 6. A kit for diagnosing Helicobacter pylori infection comprising: a) an aqueous solution having a pH of 2 to 4 containing a dose of exactly 10 to 50 mg of ¹³C-urea and a pharmacologically acceptable organic acid; b) a patient instruction sheet; and c) two sample vessels for receiving blood samples, the sample vessels being suitable for holding a strong, non-volatile acid.
 7. The diagnostic kit of claim 6 further comprising a strong, non-volatile acid in each of the two sample vessels.
 8. The diagnostic kit of claim 6, wherein the organic acid is citric acid.
 9. The diagnostic kit of claim 7, wherein the organic acid is citric acid.
 10. The diagnostic kit of claim 6 further comprising a blood sampling device.
 11. The diagnostic kit of claim 7 further comprising a blood sampling device.
 12. The diagnostic kit of claim 8 further comprising a blood sampling device.
 13. The diagnostic kit of claim 9 further comprising a blood sampling device.
 14. A kit for diagnosing Helicobacter pylori infection comprising: a) a 1^(st) container holding 10 to 50 mg of ¹³C-urea, b) a 2^(nd) container holding a solid, pharmacologically acceptable organic acid, d) a patient instruction sheet, and e) two sample vessels for receiving blood samples, the sample vessels being suitable for holding a strong, non-volatile acid.
 15. The diagnostic kit of claim 14, further comprising a strong, non-volatile acid in each of the two sample vessels.
 16. The diagnostic kit of claim 14, wherein the organic acid is citric acid.
 17. The diagnostic kit of claim 15, wherein the organic acid is citric acid.
 18. The diagnostic kit of claim 14, further comprising a blood sampling device.
 19. The diagnostic kit of claim 15, further comprising a blood sampling device.
 20. The diagnostic kit of claim 16, further comprising a blood sampling device.
 21. The diagnostic kit of claim 17, further comprising a blood sampling device. 